Evaluation the effect of thiamin deficiency on intestinal immunity of young grass carp (Ctenopharyngodon idella)

Fish & Shellfish Immunology 46 (2015) 501e515

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Evaluation the effect of thiamin deficiency on intestinal immunity of young grass carp (Ctenopharyngodon idella) Ling-Mei Wen a, Wei-Dan Jiang a, b, Yang Liu a, b, Pei Wu a, b, Juan Zhao a, Jun Jiang a, b, Sheng-Yao Kuang d, Ling Tang d, Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a, b, c, *, Lin Feng a, b, c, ** a

Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China d Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China e Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2014 Received in revised form 29 June 2015 Accepted 1 July 2015 Available online 6 July 2015

Our study explored the effect of dietary thiamin on growth and immunity (intestine, head kidney, spleen and liver) of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing six graded levels of thiamin at 0.12e2.04 mg/kg diet for 8 weeks. The percentage weight gain (PWG), feed intake and feed efficiency were lower in fish fed the 0.12 mg/kg diet. Thiamin deficiency decreased complement 3 content, lysozyme (LA) and acid phosphatase activities, mRNA levels of hepcidin and interleukin (IL) 10, elevated mRNA levels of interferon g2, tumor necrosis factor a, IL-1b and IL-8 in intestine, head kidney, spleen and liver. The mRNA levels of inhibitor protein-kBa, target of rapamycin (TOR) and NF-E2-related factor 2 (Nrf2), the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase were down-regulated, mRNA levels of myosin light-chain kinase (MLCK), IkB kinases (IKKb and IKKg), nuclear factor kB P65 (NF-kB P65) and Kelch-like-ECH-associated protein 1a (Keap1a) were up-regulated in the intestine of fish fed the thiamin-deficient diet. Additionally, thiamin deficiency decreased claudin b, c and 3, ZO-1 and occludin mRNA levels in each intestinal segment, increased claudin 12 and claudin 15a mRNA levels in distal intestine. In conclusion, thiamin deficiency decreased fish growth and immunity of intestine, head kidney, spleen and liver. The dietary thiamin requirement of young grass carp (242e742 g) based on intestinal LA activity or PWG were determined to be 1.15 or 0.90 mg/kg diet, respectively. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Thiamin deficiency Grass carp Immune response Tight junction Antioxidant capacity

1. Introduction The fish intestine is highly susceptible to pathogenic invasion [1]. To prevent pathogenic invasion, fish have developed intestinal mucosal immune system [2]. It had been demonstrated that disturbance the fish intestinal mucosal immune system resulted in an impaired immune response leading to poor growth performance

* Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China. ** Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China. E-mail addresses: [email protected] (X.-Q. Zhou), [email protected] (L. Feng). http://dx.doi.org/10.1016/j.fsi.2015.07.001 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

[3] and pathogenic invasion [2]. Therefore, maintaining a wellfunctioning intestinal mucosal immune system is of vital importance for fish [2]. Limited study had shown that nutrients deficiency, such as tryptophan [4] and valine [3] deficiency impaired the intestinal mucosal immune of fish. Thiamin is an essential vitamin for fish [5]. To date, no study has paid attention to the effect of thiamin on fish intestinal mucosal immune system. Wellers et al. [6] demonstrated that dietary thiamin deficiency resulted in decreasing the absorption of valine in rat intestine. Moreover, study from our laboratory had been revealed that dietary valine limitation impaired the intestinal mucosal immunity of grass carp [3]. The above data indicating that there may be a possible correlation between thiamin and fish intestinal mucosal immune response, which is worthy of investigation.

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L.-M. Wen et al. / Fish & Shellfish Immunology 46 (2015) 501e515

Fish intestinal mucosal immune system is broadly consisted of immune barrier and physical barrier, and the fish intestinal immune barrier mostly relies on its immune response [2]. Fish immune response is closely related to complement [7], lysozyme (LA) [8], acid phosphatase [9], antimicrobial peptides (such as hepcidin and liver expressed antimicrobial peptides 2) and cytokines including interferon g (IFN-g), tumor necrosis factor a (TNF-a), transforming growth factor-b (TGF-b), interleukin 1 (IL-1), interleukin 8 (IL-8) and interleukin 10 (IL-10) [10]. To date, studies had explored the effect of thiamin on fish growth [5], reproduction activity [11] and in vitro immune functions [12]. Additionally, in gold fish (Carassius auratus), study demonstrated that thiamin can prevent the hyperplasia of macrophages [13]. Moreover, macrophages can act to release LA [14] in fish. The above information indicating that thiamin may have effect on fish intestinal immune response, which is valuable for investigation. Furthermore, the expression of cytokines could be involved in nuclear factor kB (NFkB) and target of rapamycin (TOR) signaling pathway in activated mouse microglial cells [15]. Studies from our laboratory demonstrated that nutrients, such as valine [3] and tryptophan [4] regulated the mRNA levels of cytokines partly by affecting the expression of NF-kB and TOR in fish. However, no information has concerned about whether thiamin could modulate cytokines expression through NF-kB and TOR in fish. Langlais and Zhang [16] reported that thiamin deficiency can affect the glutamate level in rats thalamus. Moreover, glutamate can regulate the NF-kB expression level in the rat brain astrocytes [17] and the expression of mTOR in chick glia cells [18]. These observations lead to the idea that thiamin may modulate cytokines expression through NF-kB and TOR in fish, which need to be investigated. Except for the intestinal immune barrier, fish also developed physical barrier in the intestinal mucosal immune system, which is made up of intestinal epithelial cells and tight junctions (TJs) [2]. TJs is mainly composed of zonula occludens (ZO), the transmembrane proteins occludin and claudin in fish [19]. TJs play a vital role in preventing pathogen invasion in fish intestine [20]. However, there is no available information about the relationship between thiamin and TJs in fish. In rats brain, thiamin deficiency increased the endothelial nitric oxide synthase (eNOS) expression level [21]. Moreover, Beauchesne et al. [22] demonstrated that eNOS affected the expression of ZO-1 and occludin in the medial thalamus of mice. The above information suggesting that thiamin may have effect on TJs in fish, which warrants further investigation. Additionally, study had shown that the disruption of TJs is closely related to oxidative damage in human colonic Caco-2 cells [23]. Antioxidant system (including non-enzymatic compounds and antioxidant enzymes) play an important role in preventing oxidative damage [24]. The antioxidant enzymes activities are partly related to their mRNA levels in Japanese eel (Anguilla japonica) [25]. Studies revealed that NF-E2-related factor 2 (Nrf2) and its cytosolic repressor Kelch-like ECH-associated protein 1 (Keap1) play a critical role in regulating antioxidant enzymes mRNA expression in vertebrate [26,27]. Our previous studies showed that nutrient, such as tryptophan [4] regulated the mRNA levels of antioxidant enzymes partly by affecting the expression of Nrf2 and Keap1 in fish. However, no reports at present have concerned about whether thiamin could affect the mRNA expression of antioxidant enzymes via Keap1/Nrf2 signaling pathway in animal. In human brain, thiamin can affect the formation of prostaglandins [28]. Furthermore, prostaglandins up-regulated the expression of Nrf2 in multiple myeloma cells [29]. These information indicating that thiamin may partly through modulating Keap1/Nrf2 signaling pathway to affect the antioxidant capacity of fish, which is valuable for investigation. In addition to intestine, fish head kidney, spleen and liver were

also important for its immune defense [30]. To date, there is no available information about the relationship between thiamin and fish head kidney, spleen and liver immunity. Previous study showed that optimal thiamin can improve the immunity of lake trout (Salvelinus namaycush) kidney leukocytes [12]. Moreover, Wu et al. [31] revealed that the effect of choline on tissue immune response of Jian carp was vary in each tissue. Accordingly, thiamin may have effect on the immunity of fish head kidney, spleen and liver, and the effect of thiamin on their immune response may vary with tissues. However, this hypothesis awaits further investigation. Grass carp is the third biggest contributor to the world's aquaculture production [32]. Nowadays, the culture of fish depends on formulated feed which relies on the nutrients requirement of this species [33]. To date, study only investigated the dietary thiamin requirement of juvenile grass carp [5], the dietary thiamin requirement of young grass carp has not been determined. Furthermore, the nutrients requirement of fish may vary with growth stages [3] and different sensitive indices [34]. Hence, it is valuable to investigate the dietary thiamin requirement of young grass carp based on immune index and growth performance. Thus, the present study was firstly conducted to evaluate the impact of dietary thiamin deficiency on young grass carp intestinal immunity.

2. Materials and methods 2.1. Experimental design and diets The composition of the basal diet is given in Table 1. Fish meal, casein and gelatin were used as the main dietary protein sources, which are found to be limiting in thiamin. The dietary protein level was fixed at 30% according to Khan et al. [33]. The experimental diets were supplemented with thiamin nitrate (Sigma, St Louis, MO, USA) to provide graded levels of thiamin. According to the method

Table 1 Nutrients composition of the basal diet. Ingredients

g kg
1

Nutrients contenta

g kg
1

Fish meal Casein Gelatin DL-Methionine (99%) a-Starch Corn starch Fish oil Soybean oil Cellulose Ca(H2PO4)2 Vitamin premix (thiamin free)b Trace mineral premixc Thiamin premixd Choline chloride (60%) Ethoxyquin (30%)

37.50 248.10 75.00 1.40 240.00 231.20 25.00 18.90 50.00 22.40 10.00 20.00 15.00 5.00 0.50

Crude protein Crude lipid n-3 n-6 Available phosphorus

293.20 45.40 10.00 10.00 6.00

a Crude protein and crude fat were measured value. Available phosphorus, n-3 and n-6 contents were calculated according to NRC (2011). b Per kilogram of thiamin-free vitamin premix (g/kg): retinyl acetate (500,000 IU/ g), 2.40 g; cholecalciferol (500,000 IU/g), 0.40 g; DL-a-tocopherol acetate (50%), 12.54 g; menadione (23%), 0.79 g; cyanocobalamin (1%), 0.81 g; D-biotin (2%), 4.91 g; folic acid (96%), 0.40 g; ascorbyl acetate (93%), 7.16 g; niacin (99%), 2.17 g; mesoinositol (99%), 19.19 g; calcium-D-pantothenate (98%), 2.43 g; riboflavin (80%), 0.55 g; pyridoxine hydrochloride (98%), 0.59 g. All ingredients were diluted with corn starch to 1 kg. c Per kilogram of trace mineral premix (g/kg): MnSO4$H2O, 1.65 g; MgSO4$H2O, 56.20 g; FeSO4$H2O, 22.90 g; ZnSO4$H2O, 0.63 g; CuSO4$5H2O, 0.02 g; KI, 0.07 g; NaSeO3, 0.004 g. All ingredients were diluted with corn starch to 1 kg. d Thiamin premix: thiamin nitrate was added to obtain graded levels of thiamin. The final thiamin concentrations in each experimental diet were determined to be 0.12, 0.43, 0.83, 1.25, 1.62 and 2.04 mg thiamin/kg diet, respectively.

L.-M. Wen et al. / Fish & Shellfish Immunology 46 (2015) 501e515

described by Klejdus et al. [35], the final thiamin concentrations in each experimental diet were determined to be 0.12, 0.43, 0.83, 1.25, 1.62 and 2.04 mg/kg diet. All ingredients were mixed, pelleted and stored at
20  C until use as described by Jiang et al. [5]. 2.2. Fish management and feeding All experimental procedures were approved by the Animal Care Advisory Committee of Sichuan Agricultural University. After a four-week adaption period as described by Lin et al. [36], grass carp were fed with the thiamin-deficient diet for 2 weeks to reduce the body storage of thiamin according to Huang et al. [37]. At the beginning of the experiment, a total of 540 young grass carp with an initial weight of 243.24 ± 1.27 g (average body length of 24.07 ± 1.86 cm) were randomly distributed into 18 experimental cages (1.4  1.4  1.4 m3) and resulted in 30 fish in each cage. Each cage was equipped with a 100 cm diameter disc in the bottom to collect the uneaten feed according to the method of Wu et al. [38]. During the experimental period, each cage was under natural light and dark cycle. Dissolved oxygen was maintained higher than 6.0 mg/L throughout the experimental period. The water temperature was averaged at 26 ± 2  C, pH value was maintained at 7.0 ± 0.5. Fish in each cage were fed with corresponding diet for four times daily to apparent satiation for 8 weeks. The uneaten feed was collected, dried and weighed to calculate the feed intake (FI) as described by Wu et al. [38].

503

280 nm. Then, cDNA was synthesized using the PrimeScript™ RT reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions. Primers for each genes were designed according to the sequences of grass carp. The primer sequences and optimal annealing temperatures are shown in Table 2. The mRNA levels of all the genes were performed on the CFX96™ Real-Time PCR Detection System (Bio-Rad, Laboratories, Inc.) according to standard protocols of the primers. Primer amplification efficiencies were approximately 100%. According to the results of our preliminary experiment about the stability of internal control genes (data not shown), b-actin was selected to normalize the mRNA levels of all the genes. The 2
DDCT method was used to calculate the mRNA levels of all the genes according to Livak and Schmittgen [49]. 2.5. Data analysis All results were presented as means ± standard deviation (SD). All data were subjected to one-way analysis of variance (ANOVA), and then followed by Duncan's multiple-range test to determine significant differences at the level of P < 0.05 among treatments using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Parameters with significant differences were subjected to a second-degree polynomial regression analysis with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). 3. Results

2.3. Sample collection and intestinal parameters assay

3.1. Growth performance

Fish from each cage were weighted at the beginning and the end of the feeding trial. 12 h after the last feeding, 18 fish were randomly selected from each treatment, anaesthetized in a benzocaine bath according to Luo et al. [3]. Then sacrificed, quickly obtained the intestine, head kidney, spleen and liver. Frozen in liquid nitrogen and then stored at
80  C until analysis as described by Huang et al. [39]. The intestine, head kidney, spleen and liver samples were homogenized on ice in 10 volumes (w v
1) of ice-cold physiological saline and centrifuged at 6000 g at 4  C for 20 min, then the collected supernatant was stored as described by Safari et al. [40] for the related parameters analysis. The complement 3 (C3) content and LA activity were measured according to Chi et al. [7] and Liu et al. [41], respectively. Acid phosphatase activity was determined as described by Chi et al. [9]. The reactive oxygen species (ROS) production and malondialdehyde (MDA) content were determined according to Ko et al. [42] and Yang et al. [43], respectively. Protein carbonyl (PC) and reduced glutathione (GSH) contents, catalase (CAT) activity, anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacity were measured as described by Kuang et al. [44]. The total superoxide dismutase (SOD), CuZnSOD and MnSOD activities were determined as described by Lu et al. [45]. The activity of glutathione peroxidase (GPx) was analyzed according to Nugroho and Fotedar [46]. Glutathione-Stransferase (GST) and glutathione reductase (GR) activities were determined as described by Gorbi et al. [24]. The lactate and adenosine triphosphate (ATP) contents were measured by the method described by Zhou et al. [47] and Wang et al. [48], respectively.

As shown in Table 3, final body weight (FBW), percentage weight gain (PWG), feed intake (FI), feed efficiency (FE) and specific growth rate (SGR) were improved (722.11, 197.02, 605.65, 0.79 and 1.94, respectively) with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued (P > 0.05). Furthermore, using the broken-line regression analysis, the dietary thiamin requirement of young grass carp (242e742 g) based on PWG was estimated to be 0.90 mg/kg diet (Fig. 1).

2.4. Real-time quantitative PCR The total RNA of intestine, head kidney, spleen and liver were extracted using RNAiso Plus Kit (Takara, Dalian, China) according to the manufacturer's instructions followed by DNase I treatment. The quantity and quality of RNA were assessed by agarose gel electrophoresis at 1% and by spectrophotometric analysis at 260 and

3.2. The immune response related parameters in intestine The C3 contents (Table 4) in proximal intestine (PI), midintestine (MI) and distal intestine (DI) were enhanced and IFN-g2 (Fig. 4A), TNF-a (Fig. 4B), IL-1b (Fig. 4C), NF-kB P65 (Fig. 5A) and IkB kinases b (IKKb) (Fig. 5D) mRNA levels were decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. The LA activities and hepcidin (Fig. 3A) mRNA levels in PI, MI and DI were elevated with dietary thiamin levels up to 0.83, 0.83 and 1.25 mg/kg diet (P < 0.05), and then plateaued. The acid phosphatase activities in PI, MI and DI were increased (56.28, 70.33 and 89.56, respectively) with dietary thiamin levels up to 0.83, 1.25 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. Using the broken-line regression analysis, the dietary thiamin requirement of young grass carp (242e742 g) based on LA activity in DI is 1.15 mg/ kg diet (Fig. 2). The liver expressed antimicrobial peptides 2 (LEAP2) (Fig. 3B), TGF-b1 (Fig. 4E) and TOR (Fig. 5F) mRNA levels in PI, MI and DI were enhanced with dietary thiamin levels up to 0.83, 0.43 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of IL-8 (Fig. 4D) and IKKg (Fig. 5E) were decreased, IL-10 (Fig. 4F) and inhibitor protein-kBa (IkBa) (Fig. 5B) mRNA levels were increased with dietary thiamin levels up to 0.83, 0.83 and 0.43 mg/kg diet in PI, MI and DI (P < 0.05), and plateaued thereafter. Thiamin had no significant effect on IKKa (Fig. 5C) mRNA levels in PI, MI and DI of fish (P > 0.05). The lactate contents (Fig. 6A) in PI, MI and DI were decreased with dietary thiamin levels up to 1.25, 1.25 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The ATP

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Table 2 Primer sequences, thermocycling conditions and accession numbers of genes selected for analyzing by real-time PCR. Gene Hepcidin Forward Reverse LEAP-2 Forward Reverse IFN-g2 Forward Reverse TNF-a Forward Reverse IL-1b Forward Reverse IL-8 Forward Reverse TGF-b1 Forward Reverse IL-10 Forward Reverse NF-kB P65 Forward Reverse IkBa Forward Reverse IKKa Forward Reverse IKKb Forward Reverse IKKg Forward Reverse TOR Forward Reverse Claudin b Forward Reverse Claudin c Forward Reverse Claudin 3 Forward Reverse Claudin 12 Forward Reverse Claudin 15a Forward Reverse ZO-1 Forward Reverse Occludin Forward Reverse MLCK Forward Reverse CuZnSOD Forward Reverse MnSOD Forward Reverse CAT Forward Reverse

Sequences of primers

Thermocycling conditions

Accession number

50 -AGCAGGAGCAGGATGAGC-30 50 -GCCAGGGGATTTGTTTGT-30

95  C 30 s, 40 cycles of 95  C 5 s, 59.3  C 30 s and 72  C 30 s

JQ246442

50 -TGCCTACTGCCAGAACCA-30 50 -AATCGGTTGGCTGTAGGA-30

95  C 30 s, 40 cycles of 95  C 5 s, 59.3  C 30 s and 72  C 30 s

FJ390415

50 -TGTTTGATGACTTTGGGATG-30 50 -TCAGGACCCGCAGGAAGAC-30

95  C 30 s, 40 cycles of 95  C 5 s, 60.4  C 30 s and 72  C 30 s

FJ766439

50 -CGCTGCTGTCTGCTTCAC-30 50 -CCTGGTCCTGGTTCACTC-30

95  C 30 s, 40 cycles of 95  C 5 s, 58.4  C 30 s and 72  C 30 s

HQ696609

50 -AGAGTTTGGTGAAGAAGAGG-30 50 -TTATTGTGGTTACGCTGGA-30

95  C 30 s, 40 cycles of 95  C 5 s, 57.1  C 30 s and 72  C 30 s

JQ692172

50 -ATGAGTCTTAGAGGTCTGGGT-30 50 -ACAGTGAGGGCTAGGAGGG-30

95  C 30 s, 40 cycles of 95  C 5 s, 60.3  C 30 s and 72  C 30 s

JN663841

50 -TTGGGACTTGTGCTCTAT-30 50 -AGTTCTGCTGGGATGTTT-30

95  C 30 s, 40 cycles of 95  C 5 s, 55.9  C 30 s and 72  C 30 s

EU099588

50 -AATCCCTTTGATTTTGCC-30 50 -GTGCCTTATCCTACAGTATGTG-30

95  C 30 s, 40 cycles of 95  C 5 s, 61.4  C 30 s and 72  C 30 s

HQ388294

50 -GAAGAAGGATGTGGGAGATG-30 50 -TGTTGTCGTAGATGGGCTGAG-30

95  C 30 s, 40 cycles of 95  C 5 s, 62.3  C 30 s and 72  C 30 s

KJ526214

50 -TCTTGCCATTATTCACGAGG-30 50 -TGTTACCACAGTCATCCACCA-30

95  C 30 s, 40 cycles of 95  C 5 s, 62.3  C 30 s and 72  C 30 s

KJ125069

50 -GGCTACGCCAAAGACCTG-30 50 -CGGACCTCGCCATTCATA-30

95  C 30 s, 40 cycles of 95  C 5 s, 60.3  C 30 s and 72  C 30 s

KM279718

50 - GTGGCGGTGGATTATTGG-30 50 - GCACGGGTTGCCAGTTTG-30

95  C 30 s, 40 cycles of 95  C 5 s, 60.3  C 30 s and 72  C 30 s

KP125491

50 -AGAGGCTCGTCATAGTGG-30 50 -CTGTGATTGGCTTGCTTT-30

95  C 30 s, 40 cycles of 95  C 5 s, 58.4  C 30 s and 72  C 30 s

KM079079

50 -TCCCACTTTCCACCAACT-30 50 -ACACCTCCACCTTCTCCA-30

95  C 30 s, 40 cycles of 95  C 5 s, 61.4  C 30 s and 72  C 30 s

JX854449

50 -GAGGGAATCTGGATGAGC-30 50 -ATGGCAATGATGGTGAGA-30

95  C 30 s, 40 cycles of 95  C 5 s, 57.0  C 30 s and 72  C 30 s

KF193860

50 -GAGGGAATCTGGATGAGC-30 50 -CTGTTATGAAAGCGGCAC-30

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

KF193859

50 -ATCACTCGGGACTTCTA-30 50 -CAGCAAACCCAATGTAG-30

95  C 30 s, 40 cycles of 95  C 5 s, 57.0  C 30 s and 72  C 30 s

KF193858

50 -CCCTGAAGTGCCCACAA-30 50 -GCGTATGTCACGGGAGAA-30

95  C 30 s, 40 cycles of 95  C 5 s, 55.4  C 30 s and 72  C 30 s

KF998571

50 -TGCTTTATTTCTTGGCTTTC-30 50 -CTCGTACAGGGTTGAGGTG-30

95  C 30 s, 40 cycles of 95  C 5 s, 59.0  C 30 s and 72  C 30 s

KF193857

50 -CGGTGTCTTCGTAGTCGG-30 50 -CAGTTGGTTTGGGTTTCAG-30

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

KJ000055

50 -TATCTGTATCACTACTGCGTCG-30 50 -CATTCACCCAATCCTCCA-30

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

KF193855

50 -GAAGGTCAGGGCATCTCA-30 50 -GGGTCGGGCTTATCTACT-30

95  C 30 s, 40 cycles of 95  C 5 s, 53.0  C 30 s and 72  C 30 s

KM279719

50 -CGCACTTCAACCCTTACA-30 50 -ACTTTCCTCATTGCCTCC-30

95  C 30 s, 40 cycles of 95  C 5 s, 61.5  C 30 s and 72  C 30 s

GU901214

50 -ACGACCCAAGTCTCCCTA-30 50 -ACCCTGTGGTTCTCCTCC-30

95  C 30 s, 40 cycles of 95  C 5 s, 60.4  C 30 s and 72  C 30 s

GU218534

50 -GAAGTTCTACACCGATGAGG-30 50 -CCAGAAATCCCAAACCAT-30

95  C 30 s, 40 cycles of 95  C 5 s, 58.7  C 30 s and 72  C 30 s

FJ560431

L.-M. Wen et al. / Fish & Shellfish Immunology 46 (2015) 501e515

505

Table 2 (continued ) Gene GPx Forward Reverse GST Forward Reverse GR Forward Reverse Nrf2 Forward Reverse Keap1a Forward Reverse Keap1b Forward Reverse b-Actin Forward Reverse

Sequences of primers

Thermocycling conditions

Accession number

50 -GGGCTGGTTATTCTGGGC-30 50 -AGGCGATGTCATTCCTGTTC-30

95  C 30 s, 40 cycles of 95  C 5 s, 61.5  C 30 s and 72  C 30 s

EU828796

50 -TCTCAAGGAACCCGTCTG-30 50 -CCAAGTATCCGTCCCACA-30

95  C 30 s, 40 cycles of 95  C 5 s, 58.4  C 30 s and 72  C 30 s

EU107283

50 -GTGTCCAACTTCTCCTGTG-30 50 -ACTCTGGGGTCCAAAACG-30

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

JX854448

50 -CTGGACGAGGAGACTGGA-30 50 -ATCTGTGGTAGGTGGAAC-30

95  C 30 s, 40 cycles of 95  C 5 s, 62.5  C 30 s and 72  C 30 s

KF733814

50 -TTCCACGCCCTCCTCAA-30 50 -TGTACCCTCCCGCTATG-30

95  C 30 s, 40 cycles of 95  C 5 s, 63.0  C 30 s and 72  C 30 s

KF811013

50 -TCTGCTGTATGCGGTGGGC-30 50 -CTCCTCCATTCATCTTTCTCG-30

95  C 30 s, 40 cycles of 95  C 5 s, 57.9  C 30 s and 72  C 30 s

KJ729125

50 -GGCTGTGCTGTCCCTGTA-30 50 -GGGCATAACCCTCGTAGAT-30

95  C 30 s, 40 cycles of 95  C 5 s, 61.4  C 30 s and 72  C 30 s

M25013

Table 3 Treatment effects on growth performance factors of young grass carp. All data are expressed as mean ± SD (n ¼ 3). Mean values within the same row with different superscripts are significantly different (P < 0.05). IBW: initial body weight (g fish
1); FBW: final body weight (g fish
1); PWG: percent weight gain (%); FI: feed intake (g fish
1); FE: feed efficiency; SGR: specific growth rate (% day
1). PWG ¼ 100%  [FBW
IBW]/IBW), FE ¼ [FBW
IBW]/FI, SGR ¼ 100%  [ln FBW
ln IBW]/days. Dietary thiamin levels (mg kg
1 diet) 0.12 IBW FBW PWG FI FE SGR

243.11 575.00 136.50 459.83 0.72 1.54

0.43 ± ± ± ± ± ±

1.84 12.99a 3.58a 1.63a 0.03a 0.03a

243.78 621.67 155.03 501.41 0.75 1.67

0.83 ± ± ± ± ± ±

1.54 4.48b 3.20b 1.15b 0.01ab 0.02b

243.11 722.11 197.02 605.65 0.79 1.94

1.25 ± ± ± ± ± ±

1.02 11.97c 3.83c 1.35c 0.02bc 0.02c

242.44 741.67 205.93 604.85 0.83 2.00

1.62 ± ± ± ± ± ±

0.96 23.69c 10.38c 1.35c 0.04c 0.06c

243.56 733.78 201.30 604.98 0.81 1.97

2.04 ± ± ± ± ± ±

1.64 12.28c 6.31c 1.03c 0.02c 0.04c

243.44 725.56 198.04 603.62 0.80 1.95

± ± ± ± ± ±

1.26 4.29c 0.52c 1.57c 0.01c 0.03c

Regressions YPWG ¼
38.440x2 þ 115.463x þ 120.434 YFI ¼
81.937x2 þ 250.262x þ 427.169 YFE ¼
0.055x2 þ 0.161x þ 0.700 YSGR ¼
0.255x2 þ 0.765x þ 1.435

contents (Fig. 6B) were increased with dietary thiamin levels up to 1.25, 0.83 and 1.25 mg/kg diet in PI, MI and DI (P < 0.05), and plateaued thereafter.

R2 R2 R2 R2

¼ ¼ ¼ ¼

0.935 0.937 0.761 0.943

P P P P

< < < <

0.01 0.01 0.01 0.01

3.3. TJ proteins and myosin light-chain kinase (MLCK) transcript abundance in intestine The claudin b (Fig. 7A) mRNA levels in PI, MI and DI were increased with dietary thiamin levels up to 1.25, 0.43 and 0.43 mg/ kg diet (P < 0.05), and then plateaued. The mRNA levels of claudin c (Fig. 7B), claudin 3 (Fig. 7C) and ZO-1 (Fig. 7F) were increased, and MLCK (Fig. 7H) mRNA levels were decreased with dietary thiamin levels up to 0.83 mg/kg diet in each intestinal segment (P < 0.05), and plateaued thereafter. Interestingly, dietary thiamin had no significant effect on claudin 12 (Fig. 7D) and claudin 15a (Fig. 7E) mRNA levels in PI and MI (P > 0.05), whereas the mRNA levels of these genes in DI were decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. The occludin (Fig. 7G) mRNA levels in PI, MI and DI were improved with dietary thiamin levels up to 0.43, 0.83 and 0.43 mg/kg diet (P < 0.05), and then plateaued.

3.4. Antioxidant-related parameters in intestine Fig. 1. Broken-line regression analysis of percentage weight gain (PWG) of young grass carp fed graded levels of thiamin.

As shown in Fig. 8 and Table 5, reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents in

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Table 4 Treatment effects on complement 3 content (C3), lysozyme activity (LA) and acid phosphatase activity in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of young grass carp. All data are expressed as mean ± SD (n ¼ 6). Mean values within the same row with different superscripts are significantly different (P < 0.05). C3: mg/g protein; LA: U/mg protein; acid phosphatase: U/mg protein. Dietary thiamin levels (mg kg
1 diet) 0.12 C3 PI 3.75 MI 3.29 DI 4.78 LA PI 22.04 MI 25.03 DI 19.44 Acid phosphatase PI 46.37 MI 49.02 DI 72.59

0.43

0.83

1.25

1.62 4.33 ± 0.20b 4.32 ± 0.16c 6.11 ± 0.44bc

2.04

± 0.15a ± 0.20a ± 0.39a

3.82 ± 0.13a 3.65 ± 0.21b 5.75 ± 0.20b

4.32 ± 0.21b 4.19 ± 0.31c 6.42 ± 0.26c

4.37 ± 0.30b 4.33 ± 0.13c 6.21 ± 0.27c

4.32 ± 0.29b 4.29 ± 0.20c 6.18 ± 0.30c

± 1.95a ± 1.99a ± 1.32a

27.03 ± 2.43b 28.52 ± 1.58b 33.53 ± 3.14b

31.77 ± 1.96c 30.09 ± 2.76bc 36.82 ± 3.57b

30.66 ± 2.45c 31.34 ± 2.40bc 45.58 ± 4.06c

32.85 ± 2.95c 31.74 ± 2.88c 43.75 ± 3.87c

30.11 ± 2.41c 30.42 ± 2.54bc 43.40 ± 3.90c

± 1.91a ± 2.76a ± 3.88a

51.06 ± 2.69b 56.64 ± 3.06b 78.76 ± 6.41b

56.28 ± 3.01c 57.11 ± 2.63b 89.56 ± 6.60c

55.77 ± 4.06c 70.33 ± 2.70c 90.26 ± 4.73c

58.60 ± 2.39c 70.08 ± 4.57c 89.62 ± 4.78c

58.19 ± 5.15c 69.02 ± 4.32c 91.60 ± 3.49c

Regressions YC3 in MI ¼
0.545x2 þ 1.687x þ 3.082 YLA in DI ¼
11.635x2 þ 36.452x þ 16.784 Yacid phosphatase in MI ¼
6.350x2 þ 24.841x þ 45.768

R2 ¼ 0.800 R2 ¼ 0.849 R2 ¼ 0.788

P < 0.01 P < 0.01 P < 0.01

thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. CuZnSOD (Fig. 9A) and Nrf2 (Fig. 10A) mRNA levels were increased with dietary thiamin levels up to 0.43 mg/kg diet in fish intestine (P < 0.05), and plateaued thereafter. The mRNA level of CAT (Fig. 9A) was the maximum in fish fed the 1.25 mg/kg diet (P < 0.05). The MnSOD (Fig. 9A), GPx, GST and GR (Fig. 9B) mRNA levels were improved and Keap1a (Fig. 10B) mRNA level was decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. However, thiamin had no significant effect on Keap1b (Fig. 10B) mRNA level in fish intestine (P > 0.05). 3.5. Immune response-related parameters in head kidney, spleen and liver Fig. 2. Broken-line regression analysis of lysozyme (LA) activity in the distal intestine of young grass carp fed graded levels of thiamin.

intestine were decreased with dietary thiamin levels up to 0.83, 1.25 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The ASA capacity, GSH content, MnSOD and CAT activities in intestine were increased with dietary thiamin levels up to 1.25 mg/kg diet (P < 0.05), and plateaued thereafter. The AHR capacity, CuZnSOD, GPx, GST and GR activities in intestine were increased with dietary

As shown in Table 6, the C3 contents in head kidney, spleen and liver were enhanced with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. LA activities in head kidney, spleen and liver were improved with dietary thiamin levels up to 0.83, 0.83 and 1.25 mg/kg diet (P < 0.05), and then plateaued. Acid phosphatase activities were increased in head kidney, spleen and liver with dietary thiamin levels up to 0.83, 1.25 and 1.25 mg/kg diet (P < 0.05), and plateaued thereafter. The mRNA levels of hepcidin (Fig. 11A) in head kidney, spleen and liver were up-regulated with dietary thiamin levels up to 0.43, 0.83 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The LEAP-2 (Fig. 11B) mRNA levels in

Fig. 3. Effect of dietary thiamin on hepcidin (A) and liver-expressed antimicrobial peptide 2 (LEAP-2) (B) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).

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Fig. 4. Effect of dietary thiamin on IFN-g2 (A), TNF-a (B), IL-1b (C), IL-8 (D), TGF-b1 (E) and IL-10 (F) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). IFN-g2: interferon g2; TNF-a: tumor necrosis factor a; IL-1b: interleukin 1b; IL-8: interleukin 8; TGF-b1: transforming growth factor b1; IL-10: interleukin 10.

spleen and liver were up-regulated with dietary thiamin levels up to 0.43 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. However, thiamin had no significant effect on the mRNA level of LEAP-2 in head kidney (P > 0.05). The mRNA levels of IFN-g2 (Fig. 12A) in head kidney, spleen and liver were down-regulated with dietary thiamin levels up to 0.83, 0.43 and 0.43 mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of TNF-a (Fig. 12B) and IL-8 (Fig. 12D) in head kidney, spleen and liver were downregulated with dietary thiamin levels up to 0.83, 0.43 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. The IL-1b (Fig. 12C) mRNA levels were decreased and IL-10 (Fig. 12F) mRNA levels were elevated in head kidney, spleen and liver with dietary thiamin levels up to 0.83, 0.83 and 0.43 mg/kg diet (P < 0.05), and then plateaued. The TGF-b1 (Fig. 12E) mRNA levels in head kidney and spleen were up-regulated with dietary thiamin levels up to 0.83 and 0.43 mg/kg diet (P < 0.05), and plateaued thereafter. However, thiamin had no significant effect on the mRNA level of TGF-b1 in liver (P > 0.05) (Table 7). 4. Discussion Our study firstly revealed that dietary thiamin deficiency resulted in poor PWG, FI and FE of young grass carp, whereas the

PWG, FI and FE were increased with the supplementation of thiamin. Study indicated that the improvement of fish growth performance was due to the elevated FI and FE [3]. Correlation analysis showed that PWG was positively correlated with FI (r ¼ þ0.994, P < 0.01) and FE (r ¼ þ0.974, P < 0.01), indicating that the improvement of young grass carp growth may be partly due to the fact that appropriate dietary thiamin level elevated the FI and FE. In vertebrate, growth performance mostly depends on intestinal health status, which is closely associated with the intestinal mucosal immune [3]. Thus, we further investigated the effect of thiamin on intestinal immunity of young grass carp. In the current study, compared with optimal thiamin level, thiamin deficiency led to decrease C3 content, LA and acid phosphatase activities, mRNA levels of hepcidin, LEAP-2, TGF-b1 and IL10, and increase the mRNA levels of IFN-g2, TNF-a, IL-1b and IL-8 in young grass carp intestine, suggesting that dietary thiamindeficient impaired the intestinal immune response of fish. Moreover, thiamin-regulated the mRNA levels of cytokines may be associated with lactate. In activated human macrophages, lactate induced up-regulation of IL-1b and IL-8 mRNA levels [50] and down-regulation of IL-10 mRNA level in mouse macrophage-like cells [51]. In our study, the lactate content was higher in PI, MI and DI of fish fed the thiamin limitation diet. Correlation analysis

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Fig. 5. Effect of dietary thiamin on NF-kB P65 (A), IkBa (B), IKKa (C), IKKb (D), IKKg (E) and TOR (F) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). NF-kB P65: nuclear factor kB P65; IkBa: inhibitor protein-kBa; IKKa: IkB kinases a; IKKb: IkB kinases b; IKKg: IkB kinases g; TOR: target of rapamycin.

Fig. 6. Effect of dietary thiamin on lactate content (A) and adenosine triphosphate (ATP) content (B) in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).

revealed that the mRNA levels of IFN-g2, TNF-a, IL-1b and IL-8 were positively correlated with lactate content, and TGF-b1 and IL-10 mRNA levels were negatively correlated with lactate content in PI, MI and DI of young grass carp, suggesting that thiamin limitation may elevate lactate production to affect pro-inflammatory

cytokines and anti-inflammatory cytokines expression in fish. The expression of pro-inflammatory cytokines is related to NFkB signaling molecule in activated mouse microglial cells [15]. In the current study, thiamin deficiency up-regulated the mRNA levels of NF-kB P65 in PI, MI and DI of young grass carp, whereas NF-kB

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Fig. 7. Effect of dietary thiamin on claudin b (A), claudin c (B), claudin 3 (C), claudin 12 (D), claudin 15a (E), ZO-1 (F), occludin (G) and myosin light-chain kinase (MLCK) (H) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).

P65 mRNA levels were decreased with the supplementation of thiamin. Correlation analysis revealed that the mRNA levels of IFNg2, TNF-a, IL-1b and IL-8 were positively correlated with NF-kB P65 mRNA levels in each intestinal segment of young grass carp, suggesting that thiamin deficiency elevated the mRNA levels of proinflammatory cytokines may be partly related to increase the mRNA expression of NF-kB P65 in fish. Furthermore, thiamin deficiency up-regulated the mRNA levels of NF-kB P65 may be partly involved in its upstream signaling molecules, such as TOR. Zhong et al. [15] reported that up-regulation of TOR suppressed the

mRNA expression of NF-kB P65 in activated mouse microglial cells. In the present study, thiamin deficiency decreased the mRNA levels of TOR in all intestinal segments, whereas the mRNA levels of TOR were elevated by optimal thiamin level. Correlation analysis demonstrated that the mRNA levels of NF-kB P65 were negatively correlated with TOR mRNA levels in young grass carp intestine, indicating that dietary thiamin limitation resulted in up-regulating the NF-kB P65 mRNA level partly by down-regulating the mRNA level of TOR in fish. Thiamin-affected the TOR mRNA level in fish may be partly related to adenosine triphosphate (ATP). Hu et al.

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Fig. 8. Effect of dietary thiamin on reactive oxygen species (ROS) production in young grass carp intestine. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).

whereas the ATP contents were increased by optimal thiamin level. Correlation analysis indicated that the TOR mRNA levels were positively correlated with the ATP contents in young grass carp intestine, suggesting that thiamin limitation resulted in downregulation of TOR expression may be partly contributed to decrease the ATP content in fish. Besides, the IkBa-mediated nuclear translocation of NF-kB P65 also plays an important role in regulating pro-inflammatory cytokines mRNA expression. In human epithelial cells, down-regulation the expression of IkBa promoted NF-kB p65 nuclear translocation, thereby up-regulating the mRNA levels of TNF-a, IL-1b and IL-8 [53]. In the present study, thiamin limitation led to down-regulate the mRNA level of IkBa in all intestinal segments of young grass carp, indicating that dietary thiamin limitation may promote NF-kB P65 nuclear translocation to elevate the mRNA levels of pro-inflammatory cytokines by downregulating the IkBa mRNA level in fish. Additionally, thiamin deficiency down-regulated the IkBa mRNA expression may be partly

Table 5 Treatment effects on antioxidant-related parameters in young grass carp intestine. All data are expressed as mean ± SD (n ¼ 6). Mean values within the same row with different superscripts are significantly different (P < 0.05). MDA (malondialdehyde) and PC (protein carbonyl): nmol/mg protein; ASA (anti-superoxide anion) and GR (glutathione reductase): U/g protein; AHR (anti-hydroxyl radical), CuZnSOD (copper/zinc superoxide dismutase), MnSOD (manganese superoxide dismutase), CAT (catalase), GPx (glutathione peroxidase) and GST (glutathione-S-transferase): U/mg protein; GSH (glutathione): mg/g protein. Dietary thiamin levels (mg kg
1 diet) 0.12 MDA PC ASA AHR GSH CuZnSOD MnSOD CAT GPx GST GR

6.82 7.01 141.79 120.43 4.50 4.32 2.03 0.90 99.56 42.66 34.14

0.43 ± ± ± ± ± ± ± ± ± ± ±

0.26d 0.39c 6.69a 8.53a 0.20a 0.12a 0.15a 0.05a 7.63a 1.59a 2.43a

5.83 5.35 148.36 132.11 5.40 4.83 2.12 1.02 108.40 45.49 36.67

0.83 ± ± ± ± ± ± ± ± ± ± ±

0.29c 0.33b 9.41ab 5.25b 0.51b 0.36b 0.17a 0.06b 4.25b 2.65ab 2.34ab

4.91 4.25 149.18 144.68 6.98 5.49 2.88 1.32 119.12 46.87 40.14

1.25 ± ± ± ± ± ± ± ± ± ± ±

0.25a 0.31a 6.95ab 3.48c 0.28c 0.18c 0.23b 0.07c 4.39c 1.01b 2.46c

5.15 4.17 152.07 144.54 7.93 5.58 3.38 1.47 118.32 47.77 37.95

1.62 ± ± ± ± ± ± ± ± ± ± ±

0.12ab 0.25a 7.04b 4.91c 0.53d 0.34c 0.28c 0.05d 8.17c 2.60b 2.22bc

5.14 4.36 152.55 143.32 7.43 5.26 3.48 1.48 115.74 48.59 38.26

2.04 ± ± ± ± ± ± ± ± ± ± ±

0.39ab 0.23a 9.00b 2.93c 0.68cd 0.13c 0.19c 0.04d 8.67bc 3.31b 3.31bc

5.27 4.56 152.83 143.16 7.87 5.32 3.40 1.51 116.45 48.21 38.53

± ± ± ± ± ± ± ± ± ± ±

0.13b 0.29a 5.40b 5.01c 0.20d 0.30c 0.26c 0.04d 8.34bc 3.11b 2.97bc

P P P P P P P

< < < < < < <

Regressions YMDA ¼ 1.140x2
3.127x þ 7.039 YPC ¼ 1.781x2
4.915x þ 7.362 YAHR ¼
14.309x2 þ 41.252x þ 116.823 YGSH ¼
1.398x2 þ 4.740x þ 3.868 YCuZnSOD ¼
0.770x2 þ 2.112x þ 4.106 YMnSOD ¼
0.505x2 þ 1.918x þ 1.648 YCAT ¼
0.224x2 þ 0.815x þ 0.774

[52] revealed that low ATP content down-regulated the mRNA level of mTOR in rat spinal cord tissues. In our study, the ATP contents were lower in PI, MI and DI of fish fed the thiamin limitation diet,

R2 R2 R2 R2 R2 R2 R2

¼ ¼ ¼ ¼ ¼ ¼ ¼

0.802 0.881 0.732 0.874 0.703 0.853 0.944

0.01 0.01 0.01 0.01 0.01 0.01 0.01

related to the IkB kinases (IKKa, IKKb and IKKg). Study had been shown that up-regulation of IKKa, IKKb and IKKg decreased the expression of IkBa in grass carp intestine [10]. In our study, dietary

Fig. 9. Effect of dietary thiamin on CuZnSOD, MnSOD and CAT (A), GPx, GST and GR (B) mRNA levels in young grass carp intestine. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). CuZnSOD: copper/zinc superoxide dismutase; MnSOD: manganese superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GST: glutathione-S-transferase; GR: glutathione reductase.

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Fig. 10. Effect of dietary thiamin on Nrf2 (A), Keap1a and Keap1b (B) mRNA levels in young grass carp intestine. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). Nrf2: NF-E2-related factor 2; Keap1a: Kelch-like-ECH-associated protein 1a; Keap1b: Kelch-like-ECH-associated protein 1b.

Table 6 Treatment effects on complement 3 content (C3), lysozyme (LA) and acid phosphatase activities in the head kidney, spleen and liver of young grass carp. All data are expressed as mean ± SD (n ¼ 6). Mean values within the same row with different superscripts are significantly different (P < 0.05). C3: mg/g protein; LA: U/mg protein; acid phosphatase: U/mg protein. Dietary thiamin levels (mg kg
1 diet) 0.12 C3 Head kidney Spleen Liver LA Head kidney Spleen Liver Acid phosphatase Head kidney Spleen Liver

0.43

0.83

1.25

1.62 6.06 ± 0.40c 5.79 ± 0.28c 3.72 ± 0.23bc

2.04

3.36 ± 0.28a 3.67 ± 0.26a 2.73 ± 0.12a

4.19 ± 0.32b 4.43 ± 0.25b 3.43 ± 0.17b

5.59 ± 0.42c 5.72 ± 0.35c 3.99 ± 0.40c

5.80 ± 0.34c 5.52 ± 0.20c 3.89 ± 0.28c

5.61 ± 0.46c 5.48 ± 0.35c 3.81 ± 0.24c

14.08 ± 1.01a 13.85 ± 0.79a 11.75 ± 0.49a

25.76 ± 2.37b 24.02 ± 1.69b 21.41 ± 1.30b

37.83 ± 2.49c 26.70 ± 2.16c 24.07 ± 2.31c

35.85 ± 2.49c 27.13 ± 2.68c 30.13 ± 1.79d

38.40 ± 3.23c 28.58 ± 1.04c 29.32 ± 1.76d

35.58 ± 1.45c 26.32 ± 1.70c 29.01 ± 2.14d

37.41 ± 1.97a 37.36 ± 2.68a 29.10 ± 2.26a

42.53 ± 3.34b 49.18 ± 3.54b 35.73 ± 2.57b

55.57 ± 3.54c 60.35 ± 3.97c 39.42 ± 2.66c

56.86 ± 3.05c 63.90 ± 2.19cd 42.83 ± 2.13d

54.09 ± 4.02c 64.73 ± 4.57d 45.39 ± 2.31d

53.01 ± 2.59c 63.02 ± 3.03cd 44.91 ± 2.82d

Regressions YC3 in head kidney ¼
1.425x2 þ 4.289x þ 2.801 YC3 in spleen ¼
1.204x2 þ 3.514x þ 3.272 YLA in head kidney ¼
13.889x2 þ 40.064x þ 10.633 YLA in spleen ¼
8.179x2 þ 22.967x þ 12.947 YLA in liver ¼
8.044x2 þ 25.647x þ 9.780 Yacid phosphatase in head kidney ¼
12.104x2 þ 34.192x þ 32.700 Yacid phosphatase in spleen ¼
13.927x2 þ 42.762x þ 33.034 Yacid phosphatase in liver ¼
5.265x2 þ 19.382x þ 27.348

R2 R2 R2 R2 R2 R2 R2 R2

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0.875 0.846 0.893 0.815 0.907 0.806 0.904 0.858

P P P P P P P P

< < < < < < < <

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

Fig. 11. Effect of dietary thiamin on hepcidin (A) and liver-expressed antimicrobial peptide 2 (LEAP-2) (B) mRNA levels in head kidney, spleen and liver of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).

thiamin deficiency elevated the mRNA expression of IKKb and IKKg, but had no significant effect on IKKa mRNA expression, implying that thiamin deficiency decreased the mRNA expression of IkBa partly by elevating the IKKb and IKKg mRNA levels rather than IKKa in fish. The fish intestinal mucosal immune also depends on

intestinal physical barrier, which is closely correlated with intestinal TJs [2]. Therefore, we next explored the effect of thiamin on intestinal TJ proteins of young grass carp. Study indicated a barrier-forming role for claudin b, c and 3, ZO1 and occludin, and decreasing the mRNA levels of these genes

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Fig. 12. Effect of dietary thiamin on IFN-g2 (A), TNF-a (B), IL-1b (C), IL-8 (D), TGF-b1 (E) and IL-10 (F) mRNA levels in head kidney, spleen and liver of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). IFN-g2: interferon g2; TNF-a: tumor necrosis factor a; IL-1b: interleukin 1b; IL-8: interleukin 8; TGF-b1: transforming growth factor b1; IL-10: interleukin 10.

resulted in impairing the TJ barrier function of gold fish gill [19]. In the present study, compared with the optimal thiamin level, thiamin deficiency down-regulated the mRNA levels of claudin b, c and 3, ZO-1 and occludin in each intestinal segment of young grass carp, indicating that thiamin deficiency impaired the fish intestinal barrier function partly by down-regulating the mRNA levels of claudin b, c and 3, ZO-1 and occludin in all intestinal segments of fish. Thiamin-affected the TJ proteins mRNA levels may be partly related to MLCK in fish. Study showed that up-regulation of MLCK decreased the mRNA expression of ZO-1 and occludin in human Caco-2 cells [54]. Results from our study firstly demonstrated that thiamin limitation up-regulated the mRNA levels of MLCK in PI, MI and DI of young grass carp. Correlation analysis revealed that the mRNA levels of ZO-1 and occludin were negatively correlated with MLCK mRNA level in each intestinal segment of young grass carp, suggesting that dietary thiamin deficiency led to down-regulate the mRNA levels of ZO-1 and occludin partly by up-regulating the mRNA level of MLCK in fish. Additionally, thiamin deficiency elevated the mRNA level of MLCK may be partly due to the upregulation of pro-inflammatory cytokines mRNA levels. Studies

had been shown that TNF-a [55] and IL-1b [56] can up-regulate the expression of MLCK in human Caco-2 cells. According to our results, correlation analysis revealed that the mRNA levels of MLCK were positively correlated with IFN-g2, TNF-a, IL-1b and IL-8 mRNA levels in young grass carp intestine, suggesting that dietary thiamin deficiency led to elevate the mRNA level of MLCK may be partly related to the up-regulated pro-inflammatory cytokines mRNA levels in fish. Interestingly, our results showed that dietary thiamin deficiency only up-regulated the mRNA levels of claudin 12 and claudin 15a in fish DI (rather than PI and MI), whereas the mRNA levels of these genes in DI were decreased with the increasing dietary thiamin concentration. This effect may be partly attributed to the different function and status of each intestinal segment of fish. Stroband et al. [57] demonstrated that the DI of grass carp is involved in ion transport. Moreover, claudin 12 is critical for Ca2þ absorption and claudin 15 is critical for Naþ absorption [3]. Additionally, the fish DI had higher bacterial numbers, thus the DI strongly demand for a tighter barrier [20]. Furthermore, elevating the gene expression of claudin 12 in gold fish gill [19] and claudin 15 in mice intestine [58]

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Table 7 Correlation coefficient of parameters in the intestine. Independent parameters

Dependent parameters

Correlation coefficients

IFN-g2 TNF-a IL-1b IL-8 TGF-b1 IL-10 IFN-g2 TNF-a IL-1b IL-8 NF-kB P65 TOR ZO-1 Occludin MLCK

Lactate content

rPI ¼ þ0.991**; rMI ¼ þ0.967**; rDI ¼ þ0.936** rPI ¼ þ0.943**; rMI ¼ þ0.896*; rDI ¼ þ0.836* rPI ¼ þ0.987**; rMI ¼ þ0.938**; rDI ¼ þ0.915* rPI ¼ þ0.867*; rMI ¼ þ0.892*; rDI ¼ þ0.923** rPI ¼
0.993**; rMI ¼
0.972**; rDI ¼
0.911* rPI ¼
0.995**; rMI ¼
0.885*; rDI ¼
0.968** rPI ¼ þ0.995**; rMI ¼ þ0.982**; rDI ¼ þ0.983** rPI ¼ þ0.946**; rMI ¼ þ0.988**; rDI ¼ þ0.964** rPI ¼ þ0.952**; rMI ¼ þ0.994**; rDI ¼ þ0.988** rPI ¼ þ0.924**; rMI ¼ þ0.986**; rDI ¼ þ0.934** rPI ¼
0.993**; rMI ¼
0.933**; rDI ¼
0.991** rPI ¼ þ0.961**; rMI ¼ þ0.901*; rDI ¼ þ0.968** rPI ¼
0.829*; rMI ¼
0.869*; rDI ¼
0.918** rPI ¼
0.893*; rMI ¼
0.965**; rDI ¼
0.888* rPI ¼ þ0.901*; rMI ¼ þ0.960**; rDI ¼ þ0.879* rPI ¼ þ0.821*; rMI ¼ þ0.945**; rDI ¼ þ0.929** rPI ¼ þ0.970**; rMI ¼ þ0.976**;rDI ¼ þ0.931** rPI ¼ þ0.764; rMI ¼ þ0.955**; rDI ¼ þ0.972** r ¼ þ0.846* r ¼ þ0.858* r ¼ þ0.937** r ¼ þ0.988** r ¼ þ0.960** r ¼ þ0.878* r ¼ þ0.881* r ¼ þ0.947** r ¼ þ0.967** r ¼ þ0.946** r ¼ þ0.957** r ¼ þ0.979**

CuZnSOD activity MnSOD activity CAT activity GPx activity GST activity GR activity CuZnSOD MnSOD CAT GPx GST GR

NF-kB P65

TOR ATP content MLCK IFN-g2 TNF-a IL-1b IL-8 CuZnSOD mRNA MnSOD mRNA CAT mRNA GPx mRNA GST mRNA GR mRNA

Nrf2

**Correlation is significant at P < 0.01; *Correlation is significant at P < 0.05.

disrupted the barrier function. Therefore, the different mRNA expression model of claudin 12 and claudin 15a in fish PI, MI and DI may be an adaption to the intestinal function and status. Study had shown that the disruption of TJs is closely related to oxidative damage in human colonic Caco-2 cells [23]. Thereafter, we further investigated the correlation between thiamin and intestinal antioxidant capacity of young grass carp. Results from our study showed that dietary thiamin limitation elevated the ROS, MDA and PC contents, decreased ASA and AHR capacity, GSH content and CuZnSOD, MnSOD, CAT, GPx, GST and GR activities in young grass carp intestine, suggesting that thiamin deficiency decreased the intestinal antioxidant capacity of fish. To our knowledge, antioxidant enzymes activities are partly relies on their mRNA levels in Japanese eel [25]. Thus, we further investigated the impact of thiamin on mRNA levels of antioxidant enzymes in young grass carp intestine for the first time. The current study firstly showed that dietary thiamin deficiency resulted in down-regulating the CuZnSOD, MnSOD, CAT, GPx, GST and GR mRNA levels in young grass carp intestine. According to our results, correlation analysis revealed that CuZnSOD, MnSOD, CAT, GPx, GST and GR activities were positively related to their mRNA levels in young grass carp intestine, suggesting that thiamin limitation decreased the activities of antioxidant enzymes partly by down-regulating their mRNA levels in fish. The effect of thiamin on antioxidant enzymes mRNA levels may be partly related to Nrf2 in fish. Study had been shown that down-regulation of Nrf2 decreased the mRNA levels of CAT, GPx, GST and GR mRNA levels in human A549 cells [26]. Our results firstly demonstrated that thiamin deficiency decreased the mRNA level of Nrf2 in young grass carp intestine. Correlation analysis revealed that CuZnSOD, MnSOD, CAT, GPx, GST and GR mRNA levels were positively related to the mRNA level of Nrf2 in young grass carp intestine, indicating that thiamin deficiency resulted in down-regulating the mRNA levels of

antioxidant enzymes partly by decreasing the mRNA level of Nrf2 in fish. Thiamin deficiency down-regulated the antioxidant enzymes mRNA levels may also relate to the nuclear translocation of Nrf2. Keap1 is the cytosolic repressor of Nrf2, which can suppress the nuclear translocation of Nrf2 in zebrafish [27]. Furthermore, Keap1 possesses two isoforms (Keap1a and Keap1b) and down-regulation of Keap1a and Keap1b mRNA levels elevated the Nrf2-dependent antioxidant enzyme mRNA expression in zebrafish embryos [27]. In the present study, thiamin-deficient led to up-regulate the mRNA level of Keap1a in young grass carp intestine, but had no significant effect on Keap1b mRNA level, suggesting that thiamin deficiency resulted in suppressing the Nrf2-dependent antioxidant enzymes mRNA expression partly by up-regulating the mRNA level of Keap1a rather than Keap1b in fish. Our results showed that the effect of thiamin on C3 content, LA and acid phosphatase activities, the mRNA expression model of hepcidin, IFN-g2, TNF-a, IL-1b, IL-8 and IL-10 were similar in young grass carp intestine, head kidney, spleen and liver. However, the effect of thiamin on the mRNA expression model of LEAP-2 and TGF-b1 in young grass carp intestine, head kidney, spleen and liver were different. Results from our study demonstrated that thiamin limitation down-regulated the mRNA levels of LEAP-2 in young grass carp intestine, spleen and liver, but had no significant effect on its mRNA level in head kidney. This result may be partly attributed to the thiamin content difference in each tissue. Fitzsimons et al. [59] showed that thiamin content was low in head kidney of coho salmon (Oncorhynchus kisutch). Thus, the different mRNA expression model of LEAP-2 in fish head kidney may be partly related to the low thiamin content in fish head kidney. Moreover, thiamin deficiency decreased the mRNA levels of TGF-b1 in young grass carp intestine, head kidney and spleen, but had no significant effect on its mRNA level in liver. This result may be partly attributed to the low expression level in grass carp liver. Study had

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demonstrated that grass carp liver is not the main expression organ of TGF-b1 [60]. Therefore, the different mRNA expression model of TGF-b1 in fish liver may be partly related to its low expression level in fish liver. PWG and LA are often used to estimate the nutrients requirement of fish [34]. Based on PWG, the dietary thiamin requirement of young grass carp (242e742 g) was firstly estimated to be 0.90 mg/kg diet, which was lower than that based on LA (1.15 mg/kg diet). This result is similar with the study on dietary vitamin E requirement of cobia (Rachycentron canadum) [34]. 5. Conclusions In summary, four aspects of this study firstly demonstrated that (1) dietary thiamin deficiency impaired fish intestinal immune response partly by decreasing antibacterial compounds, regulating cytokines mRNA levels which may be partly involved in IKKb and IKKg (rather than IKKa), IkBa, NF-kB and TOR. (2) Dietary thiamin deficiency down-regulated the mRNA levels of claudin b, c and 3, ZO-1 and occludin in PI, MI and DI which might be ascribed to MLCK. Additionally, thiamin deficiency decreased the activities and mRNA levels of intestinal antioxidant enzymes of fish, which may be partly involved in Nrf2 and Keap1a (rather than Keap1b). (3) The effect of thiamin on immune response in fish intestine, head kidney, spleen and liver is different. Acknowledgments This research was financially supported by the National Basic Research Program of China (973 Program) (2014CB138600), National Department Public Benefit Research Foundation (Agriculture) of China (201003020), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20135103110001), Science and Technology Support Program of Sichuan Province of China (2014NZ0003), Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China (2012NC0007; 2013NC0045), the Demonstration of Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China (2015CC0011), Natural Science Foundation for Young Scientists of Sichuan Province (2014JQ0007) and Sichuan Province Research Foundation for Basic Research (2013JY0082). The authors would like to thank the personnel of these teams for their kind assistance. References [1] H. Zhang, B. Shen, H. Wu, L. Gao, Q. Liu, Q. Wang, et al., Th17-like immune response in fish mucosal tissues after administration of live attenuated Vibrio anguillarum via different vaccination routes, Fish Shellfish Immun. 37 (2014) 229e238. [2] L. Niklasson, H. Sundh, F. Fridell, G. Taranger, K. Sundell, Disturbance of the intestinal mucosal immune system of farmed Atlantic salmon (Salmo salar), in response to long-term hypoxic conditions, Fish Shellfish Immun. 31 (2011) 1072e1080. [3] J.-B. Luo, L. Feng, W.-D. Jiang, Y. Liu, P. Wu, J. Jiang, et al., The impaired intestinal mucosal immune system by valine deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating tight junction proteins transcript abundance in the intestine, Fish Shellfish Immun. 40 (2014) 197e207. [4] H.-L. Wen, L. Feng, W.-D. Jiang, Y. Liu, J. Jiang, S.-H. Li, et al., Dietary tryptophan modulates intestinal immune response, barrier function, antioxidant status and gene expression of TOR and Nrf2 in young grass carp (Ctenopharyngodon idella), Fish Shellfish Immun. 40 (2014) 275e287. [5] M. Jiang, F. Huang, Z. Zhao, H. Wen, F. Wu, W. Liu, et al., Dietary thiamin requirement of juvenile grass carp, Ctenopharyngodon idella, J. World Aquacult. Soc. 45 (2014) 461e468. [6] G. Wellers, N. Galent, J. Chevan, Influence of thiamin deficiency on intestinal absorption of essential amino acids, C. R. Soc. Biol. 164 (1970) 742e745. [7] C. Chi, B. Jiang, X.-B. Yu, T.-Q. Liu, L. Xia, G.-X. Wang, Effects of three strains of intestinal autochthonous bacteria and their extracellular products on the

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